Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.     

In vivo and in vitro sugar transport in frog intestine

In the frog intestine, both in vitro and in vivo, experiments were carried out in order to increase knowledge of the mechanism of sugar exit across the basolateral membrane of the enterocyte. The frog intestine was chosen because it lacks crypt cells and, consequently, any external fluid circuit mechanism during sugar transport can be avoided. Therefore, the sugar concentration in the absorbate collected on the serosal side is likely to be similar to that present underneath the basolateral membrane of the enterocyte. Under this condition, cell and absorbate sugar concentrations are similar; yet there is a concomitant net transintestinal sugar transport. Moreover, in in vivo experiments a net transintestinal sugar transport takes place even against a concentration difference. These results suggest that sugar exit across the basolateral membrane is not simply due to a chemically facilitated diffusion.     

High-resolution 1H NMR study of the solution structure of alamethicin

A 1H NMR study of the peptide alamethicin, which forms voltage-gated ion channels in membranes, is described. The molecule was studied in methanol as a function of temperature and pH. A complete assignment of the spectra is given, including several stereospecific assignments. Alamethicin was found to have a structure substantially similar to the crystal although, in solution, the C-terminal dipeptide adopts a somewhat extended conformation. The overall conformation was insensitive to the ionization of the side chain of the only ionizable group, Glu-18.     

Oral vaccination of skunks with raccoon poxvirus recombinants expressing the rabies glycoprotein or the nucleoprotein.

Twenty nine skunks (Mephitis mephitis) were vaccinated orally with raccoon poxvirus (RCN) recombinants: 10 with a recombinant expressing the rabies virus glycoprotein (RCNRG), 10 with RCNRG mixed with a recombinant expressing the rabies virus nucleoprotein (RCNRN) and nine with RCN alone. Rabies virus neutralizing antibodies were detected in six of the 20 skunks; five skunks (three given RCNRG, two given a mixture of recombinants) survived a rabies challenge that was lethal for nine skunks vaccinated with RCN alone.     

Substance P as a transglutaminase substrate: identification of the reaction products by fast atom bombardment mass spectrometry

Substance P was found to be an effective acyl donor substrate of transglutaminase in vitro, the reaction products having been examined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fast atom bombardment mass spectrometry. Electrophoretic experiments showed that Substance P incorporated 14C-labeled polyamines when incubated with purified guinea pig liver transglutaminase and Ca2+. Extensive use of fast atom bombardment mass spectrometry allowed to establish that: i) a 1:1 adduct Substance P-spermine is formed; ii) only a single glutamine residue out of two, i.e. Gln-5, acts as acyl donor, iii) the single lysine residue of the neuropeptide is unable to act as acyl acceptor. A direct analytical methodology to detect transglutaminase reaction products is described.     

Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts

We report the occurrence in pigeon erythrocytes of a soluble Ca2+-dependent transglutaminase (TGase) activity. The effect of the erythrocyte ghost protein modifications, determined by TGase-catalyzed reactions, on adenylate cyclase, phospholipid methyltransferase I and II activities and on the lipidic matrix fluidity of the membrane was investigated by using a purified guinea pig liver TGase preparation. The results showed a significant inhibitory effect of such modifications both on the basal and on the variously stimulated (by NaF, Gpp(NH)p alone or in the presence of 1-isoproterenol) adenylate cyclase activity. By contrast, both the phospholipid methylation and the fluidity of the lipidic matrix of the membrane were unaffected by TGase-mediated reactions. These data suggest a new possible inhibitory mechanism of the cyclic AMP synthesis which might be triggered by the enhancement of the cytosolic Ca2+ concentration.     

Purification of Tissue Forms (Amastigotes) of Trypanosoma cruzi by Immunoaffinity Chromatography1

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Scpharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.